Corn event TC1507 and methods for detection thereof

ABSTRACT

The invention provides DNA compositions that relate to transgenic insect resistant maize plants. Also provided are assays for detecting the presence of the maize TC1507 event based on the DNA sequence of the recombinant construct inserted into the maize genome and the DNA sequences flanking the insertion site. Kits and conditions useful in conducting the assays are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 10/837,105 filed Apr.30, 2004, which claims priority to U.S. Provisional Application No.60/467,772, filed May 2, 2003.

FIELD OF THE INVENTION

The present invention relates to the field of plant molecular biology,specifically the invention relates to a DNA construct for conferringinsect resistance to a plant. The invention more specifically relates toan insect resistant corn plant TC1507 and to assays for detecting thepresence of corn plant TC1507 DNA in a sample and compositions thereof.

BACKGROUND OF THE INVENTION

This invention relates to the insect resistant corn (Zea mays) plantTC1507, also referred to as maize line TC1507 or maize event TC1507, andto the DNA plant expression construct of corn plant TC1507 and thedetection of the transgene/flanking insertion region in corn plantTC1507 and progeny thereof.

Corn is an important crop and is a primary food source in many areas ofthe world. Damage caused by insect pests is a major factor in the lossof the world's corn crops, despite the use of protective measures suchas chemical pesticides. In view of this, insect resistance has beengenetically engineered into crops such as corn in order to controlinsect damage and to reduce the need for traditional chemicalpesticides. One group of genes which have been utilized for theproduction of transgenic insect resistant crops are the delta-endotoxinsfrom Bacillus thuringiensis (B. t). Delta-endotoxins have beensuccessfully expressed in crop plants such as cotton, potatoes, rice,sunflower, as well as corn, and have proven to provide excellent controlover insect pests. (Perlak, F. J et al (1990) Bio/Technology 8, 939-943;Perlak, F. J. et al. (1993) PlantMol. Biol. 22: 313-321; Fujimoto H. etal. (1993) Bio/Technology 11: 1151-1155; Tu et al. (2000) NatureBiotechnology 18: 1101-1104; PCT publication number WO 01/13731; andBing J W et al. (2000) Efficacy of Cry1F Transgenic Maize, 14^(th)Biennial International Plant Resistance to Insects Workshop, FortCollins, Colo.).

The expression of foreign genes in plants is known to be influenced bytheir location in the plant genome, perhaps due to chromatin structure(e.g., heterochromatin) or the proximity of transcriptional regulatoryelements (e.g., enhancers) close to the integration site (Weising etal., Ann. Rev. Genet 22: 421-477, 1988). At the same time the presenceof the transgene at different locations in the genome will influence theoverall phenotype of the plant in different ways. For this reason, it isoften necessary to screen a large number of events in order to identifyan event characterized by optimal expression of an introduced gene ofinterest. For example, it has been observed in plants and in otherorganisms that there may be a wide variation in levels of expression ofan introduced gene among events. There may also be differences inspatial or temporal patterns of expression, for example, differences inthe relative expression of a transgene in various plant tissues, thatmay not correspond to the patterns expected from transcriptionalregulatory elements present in the introduced gene construct. For thisreason, it is common to produce hundreds to thousands of differentevents and screen those events for a single event that has desiredtransgene expression levels and patterns for commercial purposes. Anevent that has desired levels or patterns of transgene expression isuseful for introgressing the transgene into other genetic backgrounds bysexual outcrossing using conventional breeding methods. Progeny of suchcrosses maintain the transgene expression characteristics of theoriginal transformant. This strategy is used to ensure reliable geneexpression in a number of varieties that are well adapted to localgrowing conditions.

It would be advantageous to be able to detect the presence of aparticular event in order to determine whether progeny of a sexual crosscontain a transgene of interest. In addition, a method for detecting aparticular event would be helpful for complying with regulationsrequiring the pre-market approval and labeling of foods derived fromrecombinant crop plants, for example, or for use in environmentalmonitoring, monitoring traits in crops in the field, or monitoringproducts derived from a crop harvest, as well as for use in ensuringcompliance of parties subject to regulatory or contractual terms.

It is possible to detect the presence of a transgene by any nucleic aciddetection method known in the art including, but not limited to, thepolymerase chain reaction (PCR) or DNA hybridization using nucleic acidprobes. These detection methods generally focus on frequently usedgenetic elements, such as promoters, terminators, marker genes, etc.,because for many DNA constructs, the coding region is interchangeable.As a result, such methods may not be useful for discriminating betweendifferent events, particularly those produced using the same DNAconstruct or very similar constructs unless the DNA sequence of theflanking DNA adjacent to the inserted heterologous DNA is known. Forexample, an event-specific PCR assay is described in U.S. Pat. No.6,395,485 for the detection of elite event GAT-ZM1. Accordingly, itwould be desirable to have a simple and discriminative method for theidentification of event TC1507.

SUMMARY OF THE INVENTION

This invention relates preferably to methods for producing and selectingan insect resistant monocot crop plant. More specifically, a DNAconstruct is provided that when expressed in plant cells and plantsconfers resistance to insects. According to one aspect of the invention,a DNA construct, capable of introduction into and replication in a hostcell, is provided that when expressed in plant cells and plants confersinsect resistance to the plant cells and plants. The DNA construct iscomprised of a DNA molecule named PHI8999A and it includes two transgeneexpression cassettes. The first expression cassette comprises a DNAmolecule which includes the promoter, 5′ untranslated exon, and firstintron of the maize ubiquitin (Ubi-1) gene (Christensen et al. (1992)Plant Mol. Biol. 18: 675-689 and Christensen and Quail (1996) TransgenicRes. 5: 213-218) operably connected to a DNA molecule encoding a B.t.δ-endotoxin identified as Cry1F (U.S. Pat. Nos. 5,188,960 and 6,218,188)operably connected to a DNA molecule comprising a 3′ ORF25transcriptional terminator isolated from Agrobacterium tumefaciens(Barker et al. (1983) Plant Mol. Biol. 2: 335-350). The second transgeneexpression cassette of the DNA construct comprises a DNA molecule of thecauliflower mosaic virus (CaMV) 35S promoter (Odell J. T. et al. (1985)Nature 313: 810-812; Mitsuhara et al. (1996) Plant Cell Physiol. 37:49-59) operably connected to a DNA molecule encoding a phosphinothricinacetyltransferase (PAT) gene (Wohlleben W. et al. (1988) Gene 70: 25-37)operably connected to a DNA molecule comprising a 3′ transcriptionalterminator from (CaMV) 35S (see Mitsuhara et al. (1996) Plant CellPhysiol. 37: 49-59). Plants containing the DNA construct are alsoprovided.

According to another aspect of the invention, compositions and methodsare provided for identifying a novel corn plant designated TC1507, whichmethods are based on primers or probes which specifically recognize the5′ and/or 3′ flanking sequence of TC1507. DNA molecules are providedthat comprise primer sequences that when utilized in a PCR reaction willproduce amplicons unique to the transgenic event TC1507. These moleculesmay be selected from the group consisting of:

5′-GTAGTACTATAGATTATATTATTCGTAGAG-3′ (SEQ ID NO: 1);5′-GCCATACAGAACTCAAAATCTTTTCCGGAG-3′ (SEQ ID NO: 2);5′-CTTCAAACAAGTGTGACAAA -3′ (SEQ ID NO: 23);5′-TGTGGTGTTTGTGGCTCTGTCCTAA-3′ (SEQ ID NO: 3);5′-AGCACCTTTTCATTCTTTCATATAC-3′ (SEQ ID NO: 4); 5′-GACCTCCCCA CAGGCATGATTGATC-3′ (SEQ ID NO: 5);and complements thereof. The corn plant and seed comprising thesemolecules is an aspect of this invention. Further, kits utilizing theseprimer sequences for the identification of the TC1507 event areprovided.

An additional aspect of the invention relates to the specific flankingsequences of TC1507 described herein, which can be used to developspecific identification methods for TC1507 in biological samples. Moreparticularly, the invention relates to the 5′ and/or 3′ flanking regionsof TC1507, SEQ ID NO:21 and SEQ ID NO:22, respectively, which can beused for the development of specific primers and probes. The inventionfurther relates to identification methods for the presence of TC1507 inbiological samples based on the use of such specific primers or probes.

According to another aspect of the invention, methods of detecting thepresence of DNA corresponding to the corn event TC1507 in a sample areprovided. Such methods comprise: (a) contacting the sample comprisingDNA with a DNA primer set, that when used in a nucleic acidamplification reaction with genomic DNA extracted from corn event TC1507produces an amplicon that is diagnostic for corn event TC1507; (b)performing a nucleic acid amplification reaction, thereby producing theamplicon; and (c) detecting the amplicon.

DNA molecules that comprise the novel transgene/flanking insertionregion, SEQ ID NO: 26 and SEQ ID NO: 27 and are homologous orcomplementary to SEQ ID NO: 26 and SEQ ID NO: 27 are an aspect of thisinvention.

DNA sequences that comprise the novel transgene/flanking insertionregion, SEQ ID NO:26 are an aspect of this invention. DNA sequences thatcomprise a sufficient length of polynucleotides of transgene insertsequence and a sufficient length of polynucleotides of maize genomicand/or flanking sequence from maize plant TC1507 of SEQ ID NO:26 thatare useful as primer sequences for the production of an amplicon productdiagnostic for maize plant TC1507 are included.

In addition, DNA sequences that comprise the novel transgene/flankinginsertion region, SEQ ID NO:27 are provided. DNA sequences that comprisea sufficient length of polynucleotides of transgene insert sequence anda sufficient length of polynucleotides of maize genomic and/or flankingsequence from maize plant TC1507 of SEQ ID NO:27 that are useful asprimer sequences for the production of an amplicon product diagnosticfor maize plant TC1507 are included.

According to another aspect of the invention, the DNA sequences thatcomprise at least 11 or more nucleotides of the transgene portion of theDNA sequence of SEQ ID NO:26 or complements thereof, and a similarlength of 5′ flanking maize DNA sequence of SEQ ID NO:26 or complementsthereof are useful as DNA primers in DNA amplification methods. Theamplicons produced using these primers are diagnostic for maize eventTC1507. Therefore, the invention also includes the amplicons produced byDNA primers homologous or complementary to SEQ ID NO:26.

According to another aspect of the invention, the DNA sequences thatcomprise at least 11 or more nucleotides of the transgene portion of theDNA sequence of SEQ ID NO:27 or complements thereof, and a similarlength of 3′ flanking maize DNA sequence of SEQ ID NO:27 or complementsthereof are useful as DNA primers in DNA amplification methods. Theamplicons produced using these primers are diagnostic for maize eventTC1507. Therefore, the invention also includes the amplicons produced byDNA primers homologous or complementary to SEQ ID NO:27.

More specifically, a pair of DNA molecules comprising a DNA primer set,wherein the DNA molecules are identified as SEQ ID NO: 1 or complementsthereof and SEQ ID NO: 2 or complements thereof, SEQ ID NO: 2 orcomplements thereof and SEQ ID NO: 23 or complements thereof, SEQ ID NO:3 or complements thereof and SEQ ID NO: 5 or complements thereof, SEQ IDNO: 4 or complements thereof and SEQ ID NO: 5 or complements thereof areaspects of the invention.

Further aspects of the invention include the amplicon comprising the DNAmolecules of SEQ ID NO: 1 and SEQ ID NO: 2; the amplicon comprising theDNA molecules of SEQ ID NO: 2 and SEQ ID NO: 23; the amplicon comprisingthe DNA molecules of SEQ ID NO: 3 and SEQ ID NO: 5; and the ampliconcomprising the DNA molecules of SEQ ID NO: 4 and SEQ ID NO: 5.

According to another aspect of the invention, methods of detecting thepresence of a DNA molecule corresponding to the TC1507 event in asample, such methods comprising: (a) contacting the sample comprisingDNA extracted from a corn plant with a DNA probe, molecule thathybridizes under stringent hybridization conditions with DNA extractedfrom corn event TC1507 and does not hybridize under the stringenthybridization conditions with a control corn plant DNA; (b) subjectingthe sample and probe to stringent hybridization conditions; and (c)detecting hybridization of the probe to the DNA. More specifically, amethod for detecting the presence of a DNA molecule corresponding to theTC1507 event in a sample, such methods, consisting of (a) contacting thesample comprising DNA extracted from a corn plant with a DNA probemolecule that consists of sequences that are unique to the event, e.g.junction sequences, wherein said DNA probe molecule hybridizes understringent hybridization conditions with DNA extracted from corn eventTC1507 and does not hybridize under the stringent hybridizationconditions with a control corn plant DNA; (b) subjecting the sample andprobe to stringent hybridization conditions; and (c) detectinghybridization of the probe to the DNA.

In addition, a kit and methods for identifying event TC1507 in abiological sample which detects a TC1507 specific region within SEQ IDNO: 24 are provided.

DNA molecules are provided that comprise at least one junction sequenceof TC1507 selected from the group consisting of SEQ ID NO:45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56 and 57 and complements thereof,wherein a junction sequence spans the junction between heterologous DNAinserted into the genome and the DNA from the corn cell flanking theinsertion site, i.e. flanking DNA, and is diagnostic for the TC1507event.

According to another aspect of the invention, methods of producing aninsect resistant corn plant that comprise the steps of: (a) sexuallycrossing a first parental corn line comprising the expression cassettesof the present invention, which confers resistance to insects, and asecond parental corn line that lacks insect resistance, therebyproducing a plurality of progeny plants; and (b) selecting a progenyplant that is insect resistant. Such methods may optionally comprise thefurther step of back-crossing the progeny plant to the second parentalcorn line to producing a true-breeding corn plant that is insectresistant.

The present invention provides a method of producing a corn plant thatis resistant to insects comprising transforming a corn cell with the DNAconstruct PHI8999A (SEQ ID NO:25), growing the transformed corn cellinto a corn plant, selecting the corn plant that shows resistance toinsects, and further growing the corn plant into a fertile corn plant.The fertile corn plant can be self pollinated or crossed with compatiblecorn varieties to produce insect resistant progeny.

The invention further relates to a DNA detection kit for identifyingmaize event TC1507 in biological samples. Preferably the kit of theinvention comprises a first primer which specifically recognizes the 5′or 3′ flanking region of TC1507, and a second primer which specificallyrecognizes a sequence within the foreign DNA of TC1507, or within theflanking DNA, for use in a PCR identification protocol. The inventionalso relates to a kit for identifying event TC1507 in biologicalsamples, which kit comprises a specific probe having a sequence whichcorresponds or is complementary to, a sequence having between 80% and100% sequence identity with a specific region of event TC1507.Preferably the sequence of the probe corresponds to a specific regioncomprising part of the 5′ or 3′ flanking region of event TC1507.

The methods and kits encompassed by the present invention can be usedfor different purposes such as, but not limited to the following: toidentify event TC1507 in plants, plant material or in products such as,but not limited to, food or feed products (fresh or processed)comprising, or derived from plant material; additionally oralternatively, the methods and kits of the present invention can be usedto identify transgenic plant material for purposes of segregationbetween transgenic and non-transgenic material; additionally oralternatively, the methods and kits of the present invention can be usedto determine the quality of plant material comprising maize eventTC1507. The kits may also contain the reagents and materials necessaryfor the performance of the detection method.

This invention further relates to the TC1507 corn plant or its parts,including, but not limited to, pollen, ovules, vegetative cells, thenuclei of pollen cells, and the nuclei of egg cells of the corn plantTC1507 and the progeny derived thereof. The corn plant and seed TC1507from which the DNA primer molecules of the present invention provide aspecific amplicon product is an aspect of the invention.

The foregoing and other aspects of the invention will become moreapparent from the following detailed description and accompanyingdrawing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Linear map showing the transgenic insert PHI8999A, as well asthe sequences flanking the transgenic insert.

DETAILED DESCRIPTION

The following definitions and methods are provided to better define thepresent invention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art. Definitions of common terms in molecular biologymay also be found in Rieger et al., Glossary of Genetics: Classical andMolecular, 5^(th) edition, Springer-Verlag; New York, 1991; and Lewin,Genes V, Oxford University Press: New York, 1994. The nomenclature forDNA bases as set forth at 37 CFR 1.822 is used.

As used herein, the term “comprising” means “including but not limitedto”.

As used herein, the term “corn” means Zea mays or maize and includes allplant varieties that can be bred with corn, including wild maizespecies.

As used herein, the term “TC1507 specific” refers to a nucleotidesequence which is suitable for discriminatively identifying event TC1507in plants, plant material, or in products such as, but not limited to,food or feed products (fresh or processed) comprising, or derived fromplant material.

As used herein, the terms “insect resistant” and “impacting insectpests” refers to effecting changes in insect feeding, growth, and/orbehavior at any stage of development, including but not limited to:killing the insect; retarding growth; preventing reproductivecapability; and the like.

As used herein, the terms “pesticidal activity” and “insecticidalactivity” are used synonymously to refer to activity of an organism or asubstance (such as, for example, a protein) that can be measured bynumerous parameters including, but not limited to, pest mortality, pestweight loss, pest attraction, pest repellency, and other behavioral andphysical changes of a pest after feeding on and/or exposure to theorganism or substance for an appropriate length of time. For example“pesticidal proteins” are proteins that display pesticidal activity bythemselves or in combination with other proteins.

“Coding sequence” refers to a nucleotide sequence that codes for aspecific amino acid sequence. As used herein, the terms “encoding” or“encoded” when used in the context of a specified nucleic acid mean thatthe nucleic acid comprises the requisite information to guidetranslation of the nucleotide sequence into a specified protein. Theinformation by which a protein is encoded is specified by the use ofcodons. A nucleic acid encoding a protein may comprise non-translatedsequences (e.g., introns) within translated regions of the nucleic acidor may lack such intervening non-translated sequences (e.g., as incDNA).

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. “Foreign” refers to material notnormally found in the location of interest. Thus “foreign DNA” maycomprise both recombinant DNA as well as newly introduced, rearrangedDNA of the plant. A “foreign” gene refers to a gene not normally foundin the host organism, but that is introduced into the host organism bygene transfer. Foreign genes can comprise native genes inserted into anon-native organism, or chimeric genes. A “transgene” is a gene that hasbeen introduced into the genome by a transformation procedure. The sitein the plant genome where a recombinant DNA has been inserted may bereferred to as the “insertion site” or “target site”.

As used herein, “insert DNA” refers to the heterologous DNA within theexpression cassettes used to transform the plant material while“flanking DNA” can exist of either genomic DNA naturally present in anorganism such as a plant, or foreign (heterologous) DNA introduced viathe transformation process which is extraneous to the original insertDNA molecule, e.g. fragments associated with the transformation event. A“flanking region” or “flanking sequence” as used herein refers to asequence of at least 20 base pair, preferably at least 50 base pair, andup to 5000 base pair which is located either immediately upstream of andcontiguous with or immediately downstream of and contiguous with theoriginal foreign insert DNA molecule. Transformation procedures leadingto random integration of the foreign DNA will result in transformantscontaining different flanking regions characteristic and unique for eachtransformant. When recombinant DNA is introduced into a plant throughtraditional crossing, its flanking regions will generally not bechanged. Transformants will also contain unique junctions between apiece of heterologous insert DNA and genomic DNA, or 2 pieces of genomicDNA, or 2 pieces of heterologous DNA. A “junction” is a point where 2specific DNA fragments join. For example, a junction exists where insertDNA joins flanking DNA. A junction point also exists in a transformedorganism where 2 DNA fragments join together in a manner that ismodified from that found in the native organism. “Junction DNA” refersto DNA that comprises a junction point.

As used herein, “heterologous” in reference to a nucleic acid is anucleic acid that originates from a foreign species, or, if from thesame species, is substantially modified from its native form incomposition and/or genomic locus by deliberate human intervention. Forexample, a promoter operably linked to a heterologous nucleotidesequence can be from a species different from that from which thenucleotide sequence was derived, or, if from the same species, thepromoter is not naturally found operably linked to the nucleotidesequence. A heterologous protein may originate from a foreign species,or, if from the same species, is substantially modified from itsoriginal form by deliberate human intervention.

“Regulatory sequences” refer to nucleotide sequences located upstream(5′ non-coding sequences), within, or downstream (3′ non-codingsequences) of a coding sequence, and which influence the transcription,RNA processing or stability, or translation of the associated codingsequence. Regulatory sequences may include promoters, translation leadersequences, introns, and polyadenylation recognition sequences.

“Promoter” refers to a nucleotide sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements are often referred to as enhancers. Accordingly, an “enhancer”is a nucleotide sequence that can stimulate promoter activity and may bean innate element of the promoter or a heterologous element inserted toenhance the level or tissue-specificity of a promoter. Promoters may bederived in their entirety from a native gene, or be composed ofdifferent elements derived from different promoters found in nature, oreven comprise synthetic nucleotide segments. It is understood by thoseskilled in the art that different promoters may direct the expression ofa gene in different tissues or cell types, or at different stages ofdevelopment, or in response to different environmental conditions.Promoters that cause a nucleic acid fragment to be expressed in mostcell types at most times are commonly referred to as “constitutivepromoters”. New promoters of various types useful in plant cells areconstantly being discovered; numerous examples may be found in thecompilation by Okamuro and Goldberg (1989) Biochemistry of Plants 15:1-82. It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined,nucleic acid fragments of different lengths may have identical promoteractivity.

The “translation leader sequence” refers to a nucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect numerous parameters including, processing of theprimary transcript to mRNA, mRNA stability and/or translationefficiency. Examples of translation leader sequences have been described(Turner and Foster (1995) Mol. Biotechnol. 3: 225-236).

The “3′ non-coding sequences” refer to nucleotide sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al. (1989) PlantCell 1: 671-680.

A “protein” or “polypeptide” is a chain of amino acids arranged in aspecific order determined by the coding sequence in a polynucleotideencoding the polypeptide.

A DNA construct is an assembly of DNA molecules linked together thatprovide one or more expression cassettes. The DNA construct may be aplasmid that is enabled for self replication in a bacterial cell andcontains various endonuclease enzyme restriction sites that are usefulfor introducing DNA molecules that provide functional genetic elements,i.e., promoters, introns, leaders, coding sequences, 3′ terminationregions, among others; or a DNA construct may be a linear assembly ofDNA molecules, such as an expression cassette. The expression cassettecontained within a DNA construct comprise the necessary genetic elementsto provide transcription of a messenger RNA. The expression cassette canbe designed to express in prokaryote cells or eukaryotic cells.Expression cassettes of the present invention are designed to expressmost preferably in plant cells.

The DNA molecules of the invention are provided in expression cassettesfor expression in an organism of interest. The cassette will include 5′and 3′ regulatory sequences operably linked to a coding sequence of theinvention. “Operably linked” means that the nucleic acid sequences beinglinked are contiguous and, where necessary to join two protein codingregions, contiguous and in the same reading frame. Operably linked isintended to indicate a functional linkage between a promoter and asecond sequence, wherein the promoter sequence initiates and mediatestranscription of the DNA sequence corresponding to the second sequence.The cassette may additionally contain at least one additional gene to becotransformed into the organism. Alternatively, the additional gene(s)can be provided on multiple expression cassettes or multiple DNAconstructs.

The expression cassette will include in the 5′ to 3′ direction oftranscription: a transcriptional and translational initiation region, acoding region, and a transcriptional and translational terminationregion functional in the organism serving as a host. The transcriptionalinitiation region (i.e., the promoter) may be native or analogous, orforeign or heterologous to the host organism. Additionally, the promotermay be the natural sequence or alternatively a synthetic sequence. Theexpression cassettes may additionally contain 5′ leader sequences in theexpression cassette construct. Such leader sequences can act to enhancetranslation.

It is to be understood that as used herein the term “transgenic”includes any cell, cell line, callus, tissue, plant part, or plant thegenotype of which has been altered by the presence of a heterologousnucleic acid including those transgenics initially so altered as well asthose created by sexual crosses or asexual propagation from the initialtransgenic. The term “transgenic” as used herein does not encompass thealteration of the genome (chromosomal or extra-chromosomal) byconventional plant breeding methods or by naturally occurring eventssuch as random cross-fertilization, non-recombinant viral infection,non-recombinant bacterial transformation, non-recombinant transposition,or spontaneous mutation.

A transgenic “event” is produced by transformation of plant cells with aheterologous DNA construct(s), including a nucleic acid expressioncassette that comprises a transgene of interest, the regeneration of apopulation of plants resulting from the insertion of the transgene intothe genome of the plant, and selection of a particular plantcharacterized by insertion into a particular genome location. An eventis characterized phenotypically by the expression of the transgene. Atthe genetic level, an event is part of the genetic makeup of a plant.The term “event” also refers to progeny produced by a sexual outcrossbetween the transformant and another variety that include theheterologous DNA. Even after repeated back-crossing to a recurrentparent, the inserted DNA and flanking DNA from the transformed parent ispresent in the progeny of the cross at the same chromosomal location.The term “event” also refers to DNA from the original transformantcomprising the inserted DNA and flanking sequence immediately adjacentto the inserted DNA that would be expected to be transferred to aprogeny that receives inserted DNA including the transgene of interestas the result of a sexual cross of one parental line that includes theinserted DNA (e.g., the original transformant and progeny resulting fromselfing) and a parental line that does not contain the inserted DNA.

An insect resistant TC1507 corn plant can be bred by first sexuallycrossing a first parental corn plant consisting of a corn plant grownfrom the transgenic TC1507 corn plant and progeny thereof derived fromtransformation with the expression cassettes of the present inventionthat confers insect resistance, and a second parental corn plant thatlacks insect resistance, thereby producing a plurality of first progenyplants; and then selecting a first progeny plant that is resistant toinsects; and selfing the first progeny plant, thereby producing aplurality of second progeny plants; and then selecting from the secondprogeny plants an insect resistant plant. These steps can furtherinclude the back-crossing of the first insect resistant progeny plant orthe second insect resistant progeny plant to the second parental cornplant or a third parental corn plant, thereby producing a corn plantthat is resistant to insects.

As used herein, the term “plant” includes reference to whole plants,plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, andprogeny of same. Parts of transgenic plants understood to be within thescope of the invention comprise, for example, plant cells, protoplasts,tissues, callus, embryos as well as flowers, stems, fruits, leaves, androots originating in transgenic plants or their progeny previouslytransformed with a DNA molecule of the invention and thereforeconsisting at least in part of transgenic cells, are also an aspect ofthe present invention.

As used herein, the term “plant cell” includes, without limitation,seeds, suspension cultures, embryos, meristematic regions, callustissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, andmicrospores. The class of plants that can be used in the methods of theinvention is generally as broad as the class of higher plants amenableto transformation techniques, including both monocotyledonous anddicotyledonous plants.

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. Examples of methodsof plant transformation include Agrobacterium-mediated transformation(De Blaere et al. (1987) Meth. Enzymol. 143: 277) andparticle-accelerated or “gene gun” transformation technology (Klein etal. (1987) Nature (London) 327: 70-73; U.S. Pat. No. 4,945,050,incorporated herein by reference). Additional transformation methods aredisclosed below.

Thus, isolated polynucleotides of the present invention can beincorporated into recombinant constructs, typically DNA constructs,which are capable of introduction into and replication in a host cell.Such a construct can be a vector that includes a replication system andsequences that are capable of transcription and translation of apolypeptide-encoding sequence in a given host cell. A number of vectorssuitable for stable transfection of plant cells or for the establishmentof transgenic plants have been described in, e.g., Pouwels et al.,(1985; Supp. 1987) Cloning Vectors: A Laboratory Manual, Weissbach andWeissbach (1989) Methods for Plant Molecular Biology, (Academic Press,New York); and Flevin et al., (1990) Plant Molecular Biology Manual,(Kluwer Academic Publishers). Typically, plant expression vectorsinclude, for example, one or more cloned plant genes under thetranscriptional control of 5′ and 3′ regulatory sequences and a dominantselectable marker. Such plant expression vectors also can contain apromoter regulatory region (e.g., a regulatory region controllinginducible or constitutive, environmentally- ordevelopmentally-regulated, or cell- or tissue-specific expression), atranscription initiation start site, a ribosome binding site, an RNAprocessing signal, a transcription termination site, and/or apolyadenylation signal.

It is also to be understood that two different transgenic plants canalso be mated to produce offspring that contain two independentlysegregating added, exogenous genes. Selfing of appropriate progeny canproduce plants that are homozygous for both added, exogenous genes.Back-crossing to a parental plant and out-crossing with a non-transgenicplant are also contemplated, as is vegetative propagation. Descriptionsof other breeding methods that are commonly used for different traitsand crops can be found in one of several references, e.g., Fehr, inBreeding Methods for Cultivar Development, Wilcos J. ed., AmericanSociety of Agronomy, Madison Wis. (1987).

A “probe” is an isolated nucleic acid to which is attached aconventional detectable label or reporter molecule, e.g., a radioactiveisotope, ligand, chemiluminescent agent, or enzyme. Such a probe iscomplementary to a strand of a target nucleic acid, in the case of thepresent invention, to a strand of isolated DNA from corn event TC1507whether from a corn plant or from a sample that includes DNA from theevent. Probes according to the present invention include not onlydeoxyribonucleic or ribonucleic acids but also polyamides and otherprobe materials that bind specifically to a target DNA sequence and canbe used to detect the presence of that target DNA sequence.

“Primers” are isolated nucleic acids that are annealed to acomplementary target DNA strand by nucleic acid hybridization to form ahybrid between the primer and the target DNA strand, then extended alongthe target DNA strand by a polymerase, e.g., a DNA polymerase. Primerpairs of the present invention refer to their use for amplification of atarget nucleic acid sequence, e.g., by the polymerase chain reaction(PCR) or other conventional nucleic-acid amplification methods. “PCR” or“polymerase chain reaction” is a technique used for the amplification ofspecific DNA segments (see, U.S. Pat. Nos. 4,683,195 and 4,800,159;herein incorporated by reference).

Probes and primers are of sufficient nucleotide length to bind to thetarget DNA sequence specifically in the hybridization conditions orreaction conditions determined by the operator. This length may be ofany length that is of sufficient length to be useful in a detectionmethod of choice. Generally, 11 nucleotides or more in length,preferably 18 nucleotides or more, and more preferably 22 nucleotides ormore, are used. Such probes and primers hybridize specifically to atarget sequence under high stringency hybridization conditions.Preferably, probes and primers according to the present invention havecomplete DNA sequence similarity of contiguous nucleotides with thetarget sequence, although probes differing from the target DNA sequenceand that retain the ability to hybridize to target DNA sequences may bedesigned by conventional methods. Probes can be used as primers, but aregenerally designed to bind to the target DNA or RNA and not be used inan amplification process.

Specific primers can be used to amplify an integration fragment toproduce an amplicon that can be used as a “specific probe” foridentifying event TC1507 in biological samples. When the probe ishybridized with the nucleic acids of a biological sample underconditions which allow for the binding of the probe to the sample, thisbinding can be detected and thus allow for an indication of the presenceof event TC1507 in the biological sample. Such identification of a boundprobe has been described in the art. The specific probe is preferably asequence which, under optimized conditions, hybridizes specifically to aregion within the 5′ or 3′ flanking region of the event and preferablyalso comprises a part of the foreign DNA contiguous therewith.Preferably the specific probe comprises a sequence of at least 80%,preferably between 80 and 85%, more preferably between 85 and 90%,especially preferably between 90 and 95%, and most preferably between 95and 100% identical (or complementary) to a specific region of the event.

Methods for preparing and using probes and primers are described, forexample, in Molecular Cloning: A Laboratory Manual, 2^(nd) ed., vol.1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. 1989 (hereinafter, “Sambrook et al., 1989”); CurrentProtocols in Molecular Biology, ed. Ausubel et al., Greene Publishingand Wiley-Interscience, New York, 1992 (with periodic updates)(hereinafter, “Ausubel et al., 1992”); and Innis et al., PCR Protocols:A Guide to Methods and Applications, Academic Press: San Diego, 1990.PCR primer pairs can be derived from a known sequence, for example, byusing computer programs intended for that purpose such as the PCR primeranalysis tool in Vector NTI version 6 (Informax Inc., Bethesda Md.);PrimerSelect (DNASTAR Inc., Madison, Wis.); and Primer (Version 0.5,®1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.).Additionally, the sequence can be visually scanned and primers manuallyidentified using guidelines known to one of skill in the art.

A “kit” as used herein refers to a set of reagents for the purpose ofperforming the method of the invention, more particularly, theidentification of the event TC1507 in biological samples. The kit of theinvention can be used, and its components can be specifically adjusted,for purposes of quality control (e.g. purity of seed lots), detection ofevent TC1507 in plant material, or material comprising or derived fromplant material, such as but not limited to food or feed products. “Plantmaterial” as used herein refers to material which is obtained or derivedfrom a plant.

Primers and probes based on the flanking DNA and insert sequencesdisclosed herein can be used to confirm (and, if necessary, to correct)the disclosed sequences by conventional methods, e.g., by re-cloning andsequencing such sequences. The nucleic acid probes and primers of thepresent invention hybridize under stringent conditions to a target DNAsequence. Any conventional nucleic acid hybridization or amplificationmethod can be used to identify the presence of DNA from a transgenicevent in a sample. Nucleic acid molecules or fragments thereof arecapable of specifically hybridizing to other nucleic acid moleculesunder certain circumstances. As used herein, two nucleic acid moleculesare said to be capable of specifically hybridizing to one another if thetwo molecules are capable of forming an anti-parallel, double-strandednucleic acid structure.

A nucleic acid molecule is said to be the “complement” of anothernucleic acid molecule if they exhibit complete complementarity. As usedherein, molecules are said to exhibit “complete complementarity” whenevery nucleotide of one of the molecules is complementary to anucleotide of the other. Two molecules are said to be “minimallycomplementary” if they can hybridize to one another with sufficientstability to permit them to remain annealed to one another under atleast conventional “low-stringency” conditions. Similarly, the moleculesare said to be “complementary” if they can hybridize to one another withsufficient stability to permit them to remain annealed to one anotherunder conventional “high-stringency” conditions. Conventional stringencyconditions are described by Sambrook et al., 1989, and by Haymes et al.,In: Nucleic Acid Hybridization, a Practical Approach, IRL Press,Washington, D.C. (1985), departures from complete complementarity aretherefore permissible, as long as such departures do not completelypreclude the capacity of the molecules to form a double-strandedstructure. In order for a nucleic acid molecule to serve as a primer orprobe it need only be sufficiently complementary in sequence to be ableto form a stable double-stranded structure under the particular solventand salt concentrations employed.

In hybridization reactions, specificity is typically the function ofpost-hybridization washes, the critical factors being the ionic strengthand temperature of the final wash solution. The thermal melting point(Tm) is the temperature (under defined ionic strength and pH) at which50% of a complementary target sequence hybridizes to a perfectly matchedprobe. For DNA-DNA hybrids, the Tm can be approximated from the equationof Meinkoth and Wahl (1984) Anal. Biochem. 138: 267-284: Tm=81.5°C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M is the molarityof monovalent cations, % GC is the percentage of guanosine and cytosinenucleotides in the DNA, % form is the percentage of formamide in thehybridization solution, and L is the length of the hybrid in base pairs.Tm is reduced by about 1° C. for each 1% of mismatching; thus, Tm,hybridization, and/or wash conditions can be adjusted to hybridize tosequences of the desired identity. For example, if sequences with >90%identity are sought, the Tm can be decreased 10° C. Generally, stringentconditions are selected to be about 5° C. lower than the Tm for thespecific sequence and its complement at a defined ionic strength and pH.However, severely stringent conditions can utilize a hybridizationand/or wash at 1, 2, 3, or 4° C. lower than the Tm; moderately stringentconditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10°C. lower than the Tm; low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe Tm.

Using the equation, hybridization and wash compositions, and desired Tm,those of ordinary skill will understand that variations in thestringency of hybridization and/or wash solutions are inherentlydescribed. If the desired degree of mismatching results in a Tm of lessthan 45° C. (aqueous solution) or 32° C. (formamide solution), it ispreferred to increase the SSC concentration so that a higher temperaturecan be used. An extensive guide to the hybridization of nucleic acids isfound in Tijssen (1993) Laboratory Techniques in Biochemistry andMolecular Biology—Hybridization with Nucleic Acid Probes, Part I,Chapter 2 (Elsevier, N.Y.); and Ausubel et al., eds. (1995) CurrentProtocols in Molecular Biology, Chapter 2 (Greene Publishing andWiley-Interscience, New York). See Sambrook et al. (1989) MolecularCloning: A Laboratory Manual (2d ed., Cold Spring Harbor LaboratoryPress, Plainview, N.Y.).

As used herein, a substantially homologous sequence is a nucleic acidmolecule that will specifically hybridize to the complement of thenucleic acid molecule to which it is being compared under highstringency conditions. Appropriate stringency conditions which promoteDNA hybridization, for example, 6× sodium chloride/sodium citrate (SSC)at about 45° C., followed by a wash of 2×SSC at 50° C., are known tothose skilled in the art or can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of a destabilizing agent such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. In a preferred embodiment,a nucleic acid of the present invention will specifically hybridize toone or more of the nucleic acid molecules unique to the TC1507 event orcomplements thereof or fragments of either under moderately stringentconditions.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent identity between any twosequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4: 11-17; the local homology algorithmof Smith et al. (1981) Adv. Appl. Math. 2: 482; the homology alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443-453; thesearch-for-similarity-method of Pearson and Lipman (1988) Proc. Natl.Acad. Sci. 85: 2444-2448; the algorithm of Karlin and Altschul (1990)Proc. Natl. Acad. Sci. USA 87: 2264, modified as in Karlin and Altschul(1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0); the ALIGN PLUS program (version 3.0,copyright 1997); and GAP, BESTFIT, BLAST, FASTA, and TFASTA in theWisconsin Genetics Software Package, Version 10 (available fromAccelrys, 9685 Scranton Road, San Diego, Calif. 92121, USA). Alignmentsusing these programs can be performed using the default parameters.

The CLUSTAL program is well described by Higgins and Sharp, Gene 73:237-244 (1988); Higgins and Sharp, CABIOS 5: 151-153 (1989); Corpet, etal., Nucleic Acids Research 16: 10881-90 (1988); Huang, et al., ComputerApplications in the Biosciences 8: 155-65 (1992), and Pearson, et al.,Methods in Molecular Biology 24: 307-331 (1994). The ALIGN and the ALIGNPLUS programs are based on the algorithm of Myers and Miller (1988)supra. The BLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990) supra. TheBLAST family of programs which can be used for database similaritysearches includes: BLASTN for nucleotide query sequences againstnucleotide database sequences; BLASTX for nucleotide query sequencesagainst protein database sequences; BLASTP for protein query sequencesagainst protein database sequences; TBLASTN for protein query sequencesagainst nucleotide database sequences; and TBLASTX for nucleotide querysequences against nucleotide database sequences. See, Current Protocolsin Molecular Biology, Chapter 19, Ausubel, et al., Eds., GreenePublishing and Wiley-Interscience, New York (1995). Alignment may alsobe performed manually by inspection.

To obtain gapped alignments for comparison purposes, Gapped BLAST (inBLAST 2.0) can be utilized as described in Altschul et al. (1997)Nucleic Acids Res. 25: 3389. Alternatively, PSI-BLAST (in BLAST 2.0) canbe used to perform an iterated search that detects distant relationshipsbetween molecules. See Altschul et al. (1997) supra. When utilizingBLAST, Gapped BLAST, PSI-BLAST, the default parameters of the respectiveprograms (e.g., BLASTN for nucleotide sequences, BLASTX for proteins)can be used. See, for example ncbi.nlm.nih.gov on the World Wide Web byprefacing the provided internet address with the www prefix.

As used herein, “sequence identity” or “identity” in the context of twonucleic acid or polypeptide sequences makes reference to the residues inthe two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

Regarding the amplification of a target nucleic acid sequence (e.g., byPCR) using a particular amplification primer pair, “stringentconditions” are conditions that permit the primer pair to hybridize onlyto the target nucleic-acid sequence to which a primer having thecorresponding wild-type sequence (or its complement) would bind andpreferably to produce a unique amplification product, the amplicon, in aDNA thermal amplification reaction.

The term “specific for (a target sequence)” indicates that a probe orprimer hybridizes under stringent hybridization conditions only to thetarget sequence in a sample comprising the target sequence.

As used herein, “amplified DNA” or “amplicon” refers to the product ofnucleic acid amplification of a target nucleic acid sequence that ispart of a nucleic acid template. For example, to determine whether acorn plant resulting from a sexual cross contains transgenic eventgenomic DNA from the corn plant of the present invention, DNA extractedfrom the corn plant tissue sample may be subjected to a nucleic acidamplification method using a DNA primer pair that includes a firstprimer derived from flanking sequence adjacent to the insertion site ofinserted heterologous DNA, and a second primer derived from the insertedheterologous DNA to produce an amplicon that is diagnostic for thepresence of the event DNA. Alternatively, the second primer may bederived from the flanking sequence. The amplicon is of a length and hasa sequence that is also diagnostic for the event. The amplicon may rangein length from the combined length of the primer pairs plus onenucleotide base pair to any length of amplicon producible by a DNAamplification protocol. Alternatively, primer pairs can be derived fromflanking sequence on both sides of the inserted DNA so as to produce anamplicon that includes the entire insert nucleotide sequence of thePHI8999A expression construct, see FIG. 1, approximately 6.2 Kb in size.A member of a primer pair derived from the flanking sequence may belocated a distance from the inserted DNA sequence, this distance canrange from one nucleotide base pair up to the limits of theamplification reaction, or about twenty thousand nucleotide base pairs.The use of the term “amplicon” specifically excludes primer dimers thatmay be formed in the DNA thermal amplification reaction.

Nucleic acid amplification can be accomplished by any of the variousnucleic acid amplification methods known in the art, including thepolymerase chain reaction (PCR). A variety of amplification methods areknown in the art and are described, inter alia, in U.S. Pat. Nos.4,683,195 and 4,683,202 and in PCR Protocols: A Guide to Methods andApplications, ed. Innis et al., Academic press, San Diego, 1990. PCRamplification methods have been developed to amplify up to 22 Kb ofgenomic DNA and up to 42 Kb of bacteriophage DNA (Cheng et al., Proc.Natl. Acad. Sci. USA 91: 5695-5699, 1994). These methods as well asother methods known in the art of DNA amplification may be used in thepractice of the present invention. It is understood that a number ofparameters in a specific PCR protocol may need to be adjusted tospecific laboratory conditions and may be slightly modified and yetallow for the collection of similar results. These adjustments will beapparent to a person skilled in the art.

The amplicon produced by these methods may be detected by a plurality oftechniques, including, but not limited to, Genetic Bit Analysis(Nikiforov, et al. Nucleic Acid Res. 22: 4167-4175, 1994) where a DNAoligonucleotide is designed which overlaps both the adjacent flankingDNA sequence and the inserted DNA sequence. The oligonucleotide isimmobilized in wells of a microwell plate. Following PCR of the regionof interest (using one primer in the inserted sequence and one in theadjacent flanking sequence) a single-stranded PCR product can behybridized to the immobilized oligonucleotide and serve as a templatefor a single base extension reaction using a DNA polymerase and labeledddNTPs specific for the expected next base. Readout may be fluorescentor ELISA-based. A signal indicates presence of the insert/flankingsequence due to successful amplification, hybridization, and single baseextension.

Another detection method is the Pyrosequencing technique as described byWinge (Innov. Pharma. Tech. 00: 18-24, 2000). In this method anoligonucleotide is designed that overlaps the adjacent DNA and insertDNA junction. The oligonucleotide is hybridized to a single-stranded PCRproduct from the region of interest (one primer in the inserted sequenceand one in the flanking sequence) and incubated in the presence of a DNApolymerase, ATP, sulfurylase, luciferase, apyrase, adenosine 5′phosphosulfate and luciferin. dNTPs are added individually and theincorporation results in a light signal which is measured. A lightsignal indicates the presence of the transgene insert/flanking sequencedue to successful amplification, hybridization, and single or multi-baseextension.

Fluorescence Polarization as described by Chen et al., (Genome Res. 9:492-498, 1999) is a method that can be used to detect an amplicon of thepresent invention. Using this method an oligonucleotide is designedwhich overlaps the flanking and inserted DNA junction. Theoligonucleotide is hybridized to a single-stranded PCR product from theregion of interest (one primer in the inserted DNA and one in theflanking DNA sequence) and incubated in the presence of a DNA polymeraseand a fluorescent-labeled ddNTP. Single base extension results inincorporation of the ddNTP. Incorporation can be measured as a change inpolarization using a fluorometer. A change in polarization indicates thepresence of the transgene insert/flanking sequence due to successfulamplification, hybridization, and single base extension.

Taqman® (PE Applied Biosystems, Foster City, Calif.) is described as amethod of detecting and quantifying the presence of a DNA sequence andis fully understood in the instructions provided by the manufacturer.Briefly, a FRET oligonucleotide probe is designed which overlaps theflanking and insert DNA junction. The FRET probe and PCR primers (oneprimer in the insert DNA sequence and one in the flanking genomicsequence) are cycled in the presence of a thermostable polymerase anddNTPs. Hybridization of the FRET probe results in cleavage and releaseof the fluorescent moiety away from the quenching moiety on the FRETprobe. A fluorescent signal indicates the presence of theflanking/transgene insert sequence due to successful amplification andhybridization.

Molecular Beacons have been described for use in sequence detection asdescribed in Tyangi et al. (Nature Biotech. 14: 303-308, 1996). Briefly,a FRET oligonucleotide probe is designed that overlaps the flanking andinsert DNA junction. The unique structure of the FRET probe results init containing secondary structure that keeps the fluorescent andquenching moieties in close proximity. The FRET probe and PCR primers(one primer in the insert DNA sequence and one in the flanking sequence)are cycled in the presence of a thermostable polymerase and dNTPs.Following successful PCR amplification, hybridization of the FRET probeto the target sequence results in the removal of the probe secondarystructure and spatial separation of the fluorescent and quenchingmoieties. A fluorescent signal results. A fluorescent signal indicatesthe presence of the flanking/transgene insert sequence due to successfulamplification and hybridization.

A hybridization reaction using a probe specific to a sequence foundwithin the amplicon is yet another method used to detect the ampliconproduced by a PCR reaction.

The present invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, various modifications of the invention, in addition tothose shown and described herein, will be apparent to those skilled inthe art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

The disclosure of each reference set forth herein is incorporated hereinby reference in its entirety.

EXAMPLES Example 1 Transformation of Maize by Particle Bombardment andRegeneration of Transgenic Plants Containing the Cry1F Gene

A DNA molecule of 6.2 Kb, designated PHI8999A (see FIG. 1 and SEQ IDNO:25), which includes a first transgene expression cassette comprisingthe promoter, 5′ untranslated exon, and first intron of the maizeubiquitin (Ubi-1) gene (Christensen et al. (1992) Plant Mol. Biol. 18:675-689 and Christensen and Quail (1996) Transgenic Res. 5: 213-218)operably connected to a DNA molecule encoding a Bacillus thuringiensisδ-endotoxin identified as Cry1F (U.S. Pat. Nos. 5,188,960 and 6,218,188)operably connected to a DNA molecule comprising a 3′ ORF25transcriptional terminator isolated from Agrobacterium tumefaciens(Barker et al. (1983) Plant Mol. Biol. 2: 335-350), and a secondtransgene expression cassette comprising a DNA molecule of thecauliflower mosaic virus (CaMV) 35S promoter (Odell J. T. et al. (1985)Nature 313: 810-812; Mitsuhara et al. (1996) Plant Cell Physiol. 37:49-59) operably connected to a DNA molecule encoding the selectablemarker, phosphinothricin acetyltransferase (PAT) gene (Wohlleben W. etal. (1988) Gene 70: 25-37) operably connected to a DNA moleculecomprising a 3′ transcriptional terminator from (CaMV) 35S (seeMitsuhara et al. (1996) Plant Cell Physiol. 37: 49-59) was used totransform maize embryo tissue.

B.t. Cry1F maize plants were obtained by microprojectile bombardmentusing the Biolistics® PDS-1000He particle gun manufactured by Bio-Rad,Hercules, Calif.; essentially as described by Klein et al. (1987)Nature, UK 327(6117): 70-73. Immature embryos isolated from maize ears,harvested soon after pollination were cultured on callus initiationmedium for several days. On the day of transformation, microscopictungsten particles were coated with purified PHI8999A DNA (SEQ ID NO:25)and accelerated into the cultured embryos, where the insert DNA wasincorporated into the cell chromosome. Only insert PHI8999A was usedduring transformation and no additional plasmid DNA was incorporatedinto the transformant. After bombardment, embryos were transferred tocallus initiation medium containing glufosinate as the selection agent.Individual embryos were kept physically separate during culture, and themajority of explants died on the selective medium.

Those embryos that survived and produced healthy, glufosinate-resistantcallus tissue were assigned unique identification codes representingputative transformation events, and continually transferred to freshselection medium. Plants were regenerated from tissue derived from eachunique event and transferred to the greenhouse. Leaf samples were takenfor molecular analysis to verify the presence of the transgene by PCRand to confirm expression of the Cry1F protein by ELISA. Plants werethen subjected to a whole plant bioassay using European corn borerinsects. Positive plants were crossed with inbred lines to obtain seedfrom the initial transformed plants. A number of lines were evaluated inthe field. The TC1507 event was selected from a population ofindependent transgenic events based on a superior combination ofcharacteristics, including insect resistance and agronomic performance(see Bing J W et al. (2000) Efficacy of Cry1F Transgenic Maize, 14^(th)Biennial International Plant Resistance to Insects Workshop, fortCollins, Colo., herein incorporated by reference).

Example 2 Identification of Nucleotides Comprising the Flanking Sequence5′ to the Transgenic Insert DNA in Bacillus thuringiensis Cry1F maizeline TC1507

To identify a DNA fragment that included sequence 5′ to the PHI8999Ainsert in event TC1507, Spe I restriction enzyme fragments from eventTC1507 genomic DNA were size selected on agarose gels, purified, andscreened by Southern analysis to confirm hybridization to a Cry1F probe.Following confirmation of hybridization and fragment size, the fragmentsof interest were cloned into a pBluescript II SK (+)™ cloning vector toprepare an enriched size selected plasmid based genomic DNA library. Aprobe homologous to a portion of the Cry1F gene was used to screen theplasmid library for positive clones. A positive clone was identified,purified by additional screening, and confirmed to result in a positivesignal when hybridized to the Cry1F probe. Nearly 3 Kb of the Spe Ifragment contained in the isolated positive clone was sequenced using aprimer walking approach. To initiate the first sequencing run, a primerthat binds to a known sequence in the cloning vector DNA was designed tosequence a portion of the DNA of interest. A second sequencing run overthe same region using another primer oriented in the reverse directionprovided second strand coverage. Primer walking was accomplished byrepeatedly using sequence data from previous runs to design new primersthat were then used to extend the next round of sequencing further intothe DNA of interest until the flanking sequence 5′ to the insertedtransgenic DNA in maize event TC1507 was obtained. Specific sequenceinformation is provided in Example 4.

Example 3 Confirmation of Flanking Sequence 5′ to the B.t. Cry1F MaizeLine TC1507 Insert

To confirm the 5′ flanking sequence of the B.t. Cry1F maize line TC1507insert, PCR primer pairs were designed to obtain overlapping PCRproducts extending from the 5′ flanking region into the full-lengthPHI8999A transgenic insert. PCR products were successfully amplifiedfrom B.t. Cry1F maize line TC1507 genomic DNA, isolated, and sequencedfor Region 1 through Region 6, shown in Table 1, and confirmed to matchthe previously determined sequence from the Spe I fragment, described inExample 2. However, the region from bp 2358 to bp 2829, immediatelyadjacent and 5′ to the start of the full-length insert was recalcitrantto PCR amplification and appeared to be larger than the sequenceobtained from the Spe I clone described above. The use of primer pairsflanking this region and the Advantage®-GC 2 Polymerase Mix (BDBiosciences Clontech, Palo Alto, Calif.) was successful in amplifyingPCR products from B.t. Cry1F maize line TC1507 genomic DNA forsequencing. The amplification conditions used to produce amplicons withthe Advantage®-GC 2 system are shown in Table 10. The DNA primer pairsused to confirm the sequence in the region from bp 2358 to 2829 arethose listed in SEQ ID NO:1 and SEQ ID NO:2; and SEQ ID NO:2 and SEQ IDNO:23. Sequence from this region is described in Table 1 (Regions 7a,7b, 7c, and 8).

Example 4 Event TC1507 5′ flanking sequence. A description of eachregion is provided in Table 1

Region 1 (SEQ ID NO: 28) Maize Genomic (No Significant Homology)

-   -   1 ACTAGTTTCC TAGCCCGCGT CGTGCCCCTA CCCCACCGAC GTTTATGGAA    -   51 GGTGCCATTC CACGGTTCTT CGTGGCCGCC CCTAAGGATG TAAATGGTCG    -   101 GTAAAATCCG GTAAATTTCC GGTACCGTTT ACCAGATTTT TCCAGCCGTT    -   151 TTCGGATTTA TCGGGATATA CAGAAAACGA GACGGAAACG GAATAGGTTT    -   201 TTTTTCGAAA ACGGTACGGT AAACGGTGAG ACAAACTTAC CGTCCGTTTT    -   251 CGTATTTCTC GGGAAACTCT GGTATATTCC CGTATTTGTC CCGTATTTTC    -   301 CCGACCCACG GACCTGCCAA TCAACCATCA GCCAGTCAGC CCATCCCCAC    -   351 AGCTATGGCC CATGGGGCCA TGTTGGCCAC ATGCCCACGC AACGCAAGGC    -   401 AGTAAGGCTG GCAGCCTGGC ACGCATTGAC GCATGTGGAC ACACACAGCC    -   451 GCCGCCTGTT CGTGTTTCTG TGCCGTTGTG CGAGACTGTG ACTGCGAGTG    -   501 GCGGAGTCGG CGAACGGCGA GGCGTCTCCG GAGTCTGGAC TGCGGCTGTG    -   551 GACAGCGACG CTGTGACGGC GACTCGGCGA AGCCCCAAGC TACCAAGCCC    -   601 CCAAGTCCCC ATCCATCTCT GCTTCTCTGG TCATCTCCTT CCCCTGGTCG    -   651 ATCTGCAGGC GCCAGACCG        Region 2 (SEQ ID NO: 29) Undescribed Maize Genomic Sequence        (Complement)    -   670 G CCGAAGCATC ACGAAACGCA CTAAGACCTC    -   701 GAAGGAGTCA AACCACTCCT CCGAGGCCTC GGGGGCTACA CCCGGCGGGT    -   751 GCGCTCGCGC GCACCCACCG GAACAAAATG TAACCGAGAA AGGTCGGTCC    -   801 CCTTGCAAAA AAAGTGCGAC AAAAGCCTCC AAGCGAGTAT TAACACTCAC    -   851 TTTGAGGCTC GGGGGCTAC        Region 3 (SEQ ID NO: 30) Fragment of Maize Huck-1        Retrotransposon    -   870 T GTCGGGGACC ATAATTAGGG GTACCCCCAA    -   901 GACTCCTAAT CTCAGCTGGT AACCCCCATC AGCACAAAGC TGCAAAGGCC    -   951 TGATGGGTGC GATTAAGTCA AGGCTCGGTC CACTCAAGGG ACACGATCTC    -   1001 GCCTCGCCCG AGCCCAGCCT CGGGCAAGGG CGGCCGACCC CGAGGATTCA    -   1051 CGTCTCGCCC GAGGGCCCCC TCAAGCGACG GGCACACCTT CGGCTCGCCC    -   1101 GAGGCCCATT CTTCGCCGAG AAGCAACCTT GGCCAGATCG CCACACCGAC    -   1151 CGACCGTATC GCAGGAGCAT TTAATGCGAG GATCGCCTGA CACCTTATCC    -   1201 TGACGCGCGC TCTTCAGTCG ACAGAGCCGA AGTGACCGCA ATCACTTCGC    -   1251 CGCTCCACTG ACCGACCTGA CAAGAAGACA GCGCCGCCTG CGTCGCTCCG    -   1301 ACTGCTGTGC CACTCGACAG AGTGAGGCTG ACAGCAGCCA AGTCCGGCCT    -   1351 CGGGCGCCAT AGGAAGCTCC GCCTCGCCCG ACCCTAGGGC TCGGACTCGG    -   1401 CCTCGGCTCC GGAAGACGAC GAACTACGCT TCGCCCGACC CCAGGGCTTG    -   1451 GACTCAGCCT CGGCTCCGGA AGACGACGAA TTCCGCCTCG CCCGACCCCA    -   1501 GGGCTCGGAC TCGGCCTCGG CTCCAGAAGA CGACGAACTC CGCCTCGCCC    -   1551 GACCCCAGGG CTCGGACTCA GCCTCGGCTC CGGAAGACGA CGAACTCCGC    -   1601 CTCGCCCGAC CCCAGGGCTC GGACTCAGCC TCGGCCTCAG ACGATGGTCT    -   1651 CCGCCTCGCC CGACCCGGGG CTCGGACTCG A        Region 4 (SEQ ID NO: 31) Fragment of cry1F Gene    -   1682 CCTTTCTAT CGGACCTTGT    -   1701 CAGATCCTGT CTTCGTCCGA GGAGGCTTTG GCAATCCTCA CTATGTACTC    -   1751 GGTCTTAGGG GAGTGGCCTT TCAACAAACT GGTACGAATC ACACCCGCAC    -   1801 ATTCAGGAAC TCCGGGACCA TTGACTCTCT AGATGAGATA CCACCTCAAG    -   1851 ACAACAGCGG CGCACCTTGG AATGACTACT CCCATGTGCT GAATCATGTT    -   1901 ACCTTTGTGC GCTGGCCAGG TGAGATCTCA GGTTCCGACT CATGGAGAGC    -   1951 ACCAATGTTC TCTTGGACGC ATCGTAGCGC TACCCCCACA AACACCATTG    -   2001 ATCCAGAGAG AATCAC        Region 5 (SEQ ID NO: 32) Fragment of Maize Chloroplast rpoC2        Gene    -   2017 TCAT TCTTCAAGAA CTGCATATCT TGCCGAGATC    -   2051 CTCATCCCTA AAGGTACTTG ACAATAGTAT TATTGGAGTC GATACACAAC    -   2101 TCACAAAAAA TACAAGAAGT CGACTAGGTG GATTGGTCCG AGTGAAGAGA    -   2151 AAAAAAAGCC ATACAGAACT CAAAATCTTT TCCGGAGATA TTCATTTTCC    -   2201 TGAAGAGGCG GATAAGATAT TAGGTGGCAG TTTGATACCA CCAGAAAGAG    -   2251 AAAAAAAAGA TTCTAAGGAA TCAAAAAAAA GGAAAAATTG GGTTTATGTT    -   2301 CAACGGAAAA AATTTCTCAA AAGCAAGGAA AAGTATT        Region 6 (SEQ ID NO: 33) Fragment of Maize Chloroplast or        ubiZM1(2) Promoter    -   2338 GTG GCTATTTATC    -   2351 TATC        Nucleotides 2355-2358 (CGT) Connect Region 6 to Region 7a.        Region 7a (SEQ ID NO: 34) Fragment of Pat Gene    -   2358 GCA GCTGATATGG CCGCGGTTTG TGATATCGTT AACCATTACA    -   2401 TTGAGACGTC TACAGTGAAC TTTAGGACAG AGCCACAAAC ACCACAAGAG    -   2451 TGGATTGATG ATCTAGAGAG GTTGCAAGAT AGATACCCTT GGTTGGTTGC    -   2501 TGAGGTTGAG GGTGTTGTGG CTGGTATTGC TTACGCTGGG CCCTGGAAGG    -   2551 CTAGGAAC        Region 7b (SEQ ID NO: 35) Fragment of Pat Gene (Complement)    -   2559 CC TCAACCTCAG CAACCAACCA ATGGTATCTA TCTTGCAACC    -   2601 TCTCTAGATC ATCAATCCAC TCTTGTGGTG TTTGTGGCTC TGTCCTAAAG    -   2651 TTCACTGTAG ACGTCTCAAT GTAATGGTTA ACGATATCAC AAACCG        Region 7c (SEQ ID NO: 36) Fragment of cry1F Gene (Complement)    -   2697 AGAG    -   2701 AAGAGGGATC T        Region 8 (SEQ ID NO: 37) Fragment of Polylinker    -   2712 CGAAGCTTC GGCCGGGGCC CATCGATATC CGCGGGCATG    -   2751 CCTGCAGTGC AGCGTGACCC GGTCGTGCCC CTCTCTAGAG ATAATGAGCA    -   2801 TTGCATGTCT AAGTTATAAA AAATTACCA        Region 9 (SEQ ID NO: 25) Full-Length Insert of PHI8999A

Example 5 Description of the Flanking Sequence 5′ to the Insert in MaizeEvent TC1507

In order to more fully describe the event TC1507 5′ flanking sequence,homology searching was done against the GenBank public databases(release 122, 2/01) using the Basic Local Alignment Search Tool (BLAST).The BLAST program performs sequence similarity searching and isparticularly useful for identifying homologs to an unknown sequence. Inaddition to searching the public databases, pairwise alignments wereperformed using AlignX (InforMax Inc., Bethesda, Md.) to look forhomology between the maize event TC1507 flanking sequence and thePHI8999A transgenic insert. The results of these homology searches arepresented in Table 1. The TC1507 5′ flanking sequence is numbered withbase 1 being the furthest 5′ to the insert and base 2830 at the startingpoint of the full-length PHI8999A transgenic insert (see FIG. 1). Thepercent identity values indicate the percentage of identical matchesacross the length of the sequences analyzed.

In most cases, similarity searching with the event TC1507 5′ flankingsequence resulted in a match to one unique sequence based on a very highpercent identity value. Those sequences are identified in Table 1. Inaddition, there are two regions in the TC1507 5′ DNA flanking sequencewith high similarity to more than one known sequence. In regions870-1681 and 2338-2354, the percent identity scores with both sequencefragments are sufficiently high that a single match (homolog) cannot bedetermined. The two possible homologs for each of these regions areindicated in Table 1.

Highly similar sequences were identified for all but the first 669 basepairs of sequence. Generally, the results of similarity searchingindicate high homology with maize genomic sequences 5′ to base 1681. Theregion from base 1682 to the start of the PHI8999A insert at position2830 contains some fragments associated with the transformation event.

TABLE 1 Sequence summary for event TC1507 insert Location in SEQLocation in ID Size % homologous Region NO:24 bp Identity Homologsequence Description  1 1-669 669 N/A¹ N/A N/A No significant homologydetected  2 670-869 200 90.5 AF123535 52432-52632 Undescribed(complement) maize genomic sequence  3 870-1681 812 89.4 AF050439 1-801Fragment of maize Huck-1 retrotransposon 5′ LTR² 86.6 AF050438 1-797Fragment of maize Huck-1 retro- transposon 3′ LTR  4 1682- 335 100.0PHI8999A 3149-3483 Fragment of 2016 cry1F gene  5 2017- 321 100.0 X8656329429-29749 Fragment of 2337 maize chloroplast rpoC2 gene (RNApolymerase beta-2 subunit)  6 2338- 17 100.0 X86563 97643-97659 Fragmentof 2354 maize chloroplast trnI gene (tRNA- Ile) 82.4 PHI8999A 182-197Fragment of maize ubiZM1(2) promoter  7a 2358- 201 100.0 PHI8999A5320-5475 Fragment of pat 2558 gene  7b 2559- 138 99 PHI8999A 5336-5518Fragment of pat 2696 (complement) gene  7c 2697- 15 100.0 PHI8999A2544-2558 Fragment of 2711 (complement) cry1F gene  8 2712- 118 100.0PHI8999A 36-153 Fragment of 2829 polylinker region (bases 36-80) andubiZM1(2) promoter (bases 81-153)  9 2830- 6186 100.0 PHI8999A 11-6196Full-length insert 9015 of PHI8999A 10 9016- 550 100.0 PHI8999A3906-4456 Inverted ORF25 9565 (complement) terminator 11 9566- 128 100.0NC_001666 121851- Fragment of 9693 121978 maize chloroplast (complement)rps12 rRNA (23S & ribosomal RNA) 100759- 100886 12 9696- 392 99NC_001666 17091-17483 Fragment of 10087 (complement) maize chloroplastgenome 13 10088- 188 99 PHI8999A 5333-5520 Fragment of pat 10275(complement) gene 14 10278- 81 100 NC_001666 137122- Fragment of 10358137202 maize chloroplast (complement) “ORF241” - hypothetical proteingene 15 10359- 254 N/A¹ N/A N/A No significant 10612 homology detected16 10613- 749 N/A¹ N/A N/A No description 11361 available ¹N/A; notapplicable ²LTR; long terminal repeat

Example 6 Confirmation of the Presence of Regions 1, 2, and 3 in anUnmodified Control Corn Line

PCR analysis was used to determine if Regions 1, 2, and 3 (Table 1) inthe 5′ flanking region of Event TC1507 are present in an unmodifiedcontrol corn line used for transformation to produce maize event TC1507and thus represents a border with corn genomic DNA. Nine different PCRanalyses were carried out on genomic DNA prepared from TC1507 and theunmodified control corn line Hi-II (see Armstrong (1994) The MaizeHandbook, ed. Freeling and Walbot, Springer-Verlag, New York, pp.663-671, for information on Hi-II) as outlined in Table 2 using theprimer sequences shown in Table 3. Two reactions were designed toamplify DNA within Region 1 of the 5′ flanking region from bp 25 to 324(Reaction A —300 bp amplicon); and from bp 25 to 480 (Reaction B —456 bpamplicon). The expected amplicons were present in both the Hi-IIunmodified corn line and in maize event TC1507. One PCR primer pair,Reaction C, spanned Region 2 to Region 3 of the 5′ flanking region frombp 759 to 1182 (424 bp amplicon) and again produced PCR products of theexpected size in both Hi-II and TC1507. Reaction D, spanned Region 1 toRegion 3 of the 5′ flanking region from bp 415 to 1182 (768 bp amplicon)and again produced PCR products of the expected size in both Hi-II andTC1507. Reactions E and F were designed as specific primer pairs for thepat gene region of the full-length insert of PHI8999A in TC1507 and thusan amplicon in the unmodified Hi-II corn line is not expected. Theresults indicate that both Reactions E and F are specific for a maizeline transformed with a pat gene region and produce the expectedamplicon, whereas no amplicon was produced in the unmodified Hi-II cornline. Reaction G was also designed as a primer pair that would producean amplicon of 366 bp in the maize event TC1507 and no amplicon in theunmodified Hi-II corn line.

Reactions H and I were designed as specific primer pairs for TC1507 thatwould span the end of the transgenic insert into the 5′ flanking region.In both Reactions H and I, the reverse primer was located in theubiquitin promoter region of the full-length PHI8999A insert (Region 9in Table 1) and the forward primer was located in Region 5, the rpoC2gene fragment (see Table 1). Reaction H and Reaction I both produced anamplicon in maize line TC1507 and did not produce an amplicon in theunmodified control corn line. These results indicate that both ReactionsH and I are specific for the TC1507 event.

The PCR results show that the undescribed sequence (Region 1) is presentin the unmodified corn line Hi-II and that Regions 1, 2 and 3, arecontiguous in the unmodified corn line Hi-II. The DNA sequencesamplified in Reactions A, B, C, and D are not unique to the 5′ flankingregion of maize event TC1507 but are also present in the unmodified cornline Hi-II.

TABLE 2 PCR reactions for sequence 5′ to the PHI8999A insert in maizeevent TC1507 and for regions within the full-length insert of PHI8999Ain maize event TC1507 Amplicon PCR Region in TC1507 Amplicon presentAmplicon Amplicon flanking sequence present in maize Reaction LocationSize (bp) or PHI8999A insert In Hi-II line TC1507 A 25-324 bp 300 Region1 Yes Yes in TC1507 flanking sequence B 25-480 bp 456 Region 1 Yes Yesin TC1507 flanking sequence C 759-1182 bp 424 Region 2 to Region 3 YesYes in TC1507 flanking sequence D 415-1182 bp 768 Region 1 to Region 3Yes Yes in TC1507 5′ flanking sequence E 4750-5794 bp 1045 Region 9 (infull- No Yes Not Unique in PHI8999A length insert of to TC1507 PHI8999A35S promoter to pat gene) F 4827-5308 bp 482 Region 9 (in full- No YesNot Unique in PHI8999A length insert of to TC1507 PHI8999A 35S promoterto pat gene) G cry1F sequence 366 Spans 335 bp No Yes Detects cry1F in5′ flanking cry1F sequence in fragment in sequence and in 5′ flankingsequence 5′ flanking full-length insert and same sequence in region ofPHI8999A the full-length insert H 2158 bp in Region 5 912 Region 5 toRegion 9 No Yes Unique to (rpoC2 gene fragment) Unique to InsertionTC1507 to 3069 bp in Region 9 Event (full-length insert of [SPANS UNIQUEPHI8999A) JUNCTION REGIONS] I 2158 bp in Region 5 844 Region 5 to Region9 No Yes Unique (rpoC2 gene fragment) Unique to Insertion TC1507 to 3001bp in Region 9 Event (full-length insert [SPANS UNIQUE of PHI8999A)JUNCTION REGIONS]

TABLE 3 PCR primers for sequence 5′ to the PHI8999A insert in TC1507 andfor regions within the full-length insert of PHI8999A in maize eventTC1507 Amplicon Primer Sequences Reaction Size (bp) Primer Pair 5′ to 3′A 300 SEQ ID NO:10 CCCCTACCCCACCGACGTTTAT SEQ ID NO:11TTGATTGGCAGGTCCGTGGGTC B 456 SEQ ID NO:10 CCCCTACCCCACCGACGTTTAT SEQ IDNO:12 CACAACGGCACAGAAACACGAA C 424 SEQ ID NO:13 GCGCACCCACCGGAACAAAATGSEQ ID NO:14 TCCTCGCATTAAATGCTCCTGC D 768 SEQ ID NO:15CCTGGCACGCATTGACGCATGT SEQ ID NO:14 TCCTCGCATTAAATGCTCCTGC E 1045 SEQ IDNO:6  TAGAGGACCTAACAGAACTCGCCGT SEQ ID NO:7  GAGCTGGCAACTCAAAATCCCTTT F482 SEQ ID NO:8  AAAATCTTCGTCAACATGGTGGAGC SEQ ID NO:9 TAATCTCAACTGGTCTCCTCTCCGG G 366 SEQ ID NO:19 GGCTCGGACTCGACCTTTCTAT SEQID NO:20 GCAGTTCTTGAAGAATGAGTGA H 912 SEQ ID NO:1 GTAGTACTATAGATTATATTATTCGTAGAG SEQ ID NO:2 GCCATACAGAACTCAAAATCTTTTCCGGAG I 844 SEQ ID NO:2 GCCATACAGAACTCAAAATCTTTTCCGGAG SEQ ID NO:23 CTTCAAACAAGTGTGACAAA

Example 7 Flanking Sequence 3′ to Inserted Transgenic DNA in Maize EventTC1507

Two separate PCR approaches were used to extend the length of thesequence information 3′ to the full-length PHI8999A insert in maizeevent TC1507. In the first approach PCR primer pairs were designed toamplify a product that spanned the junction between the full-lengthinsert and the inverted ORF25 terminator, see FIG. 1 for a depiction ofthe inverted ORF25 terminator. A forward primer was located at the endof the full-length PHI8999A insert and a series of reverse primers werelocated at 100 bp intervals in the inverted sequence. In this manner thelength of the inverted fragment present in the maize event TC1507 couldbe determined within a 100 bp region based on the successful PCRreactions. This method indicated the inverted fragment contained themajority of the ORF25 terminator but no Cry1F sequence. PCR fragmentswere isolated and sequenced from this region.

In the second approach PCR primers were designed to walk out into theflanking DNA sequence from the inverted ORF25 terminator region asdetermined in the PCR experiment described above. Genomic DNA isolatedfrom two to three individual plants of event TC1507 and an unmodifiedcontrol corn line was digested with various restriction enzymes and thenligated to adaptors specific for the restriction enzyme used fordigestion (Universal Genome Walker™ Kit, Clontech Laboratories, Inc. andDevon et al. (1995) Nucleic Acids Res. 23: 1644-1645). Primary PCR wascarried out using an ORF25 terminator specific primer and a primerhomologous to the adaptor sequence ligated onto the digested DNA. Inorder to increase the specificity of the reaction a nested secondary PCRwas performed again with another ORF25 terminator specific primer and asecondary primer homologous to the adaptor sequence with the secondaryprimers being internal to the respective primers used in the primaryPCR. Products produced by the nested PCR were analyzed by agarose gelelectrophoresis and fragments unique to TC1507 DNA samples were isolatedand sequenced. Fragments were amplified from both the ORF25 terminatorcontained within the full-length insert and from the targeted (inverted)ORF25 terminator on the 3′ end of the full-length PHI8999A insert.Fragments from the full-length insert were of a predicted size based onthe knowledge of the restriction enzyme sites located in the full-lengthinsert. Fragments produced from the 3′ inverted ORF25 terminatorappeared as fragments of unexpected size. Sequence analysis of amplifiedfragments from the 3′ inverted ORF25 terminator resulted in flanking DNAsequence of 1043 bp. Resultant sequence from the above series of genomewalking experiments was used to design additional primers to walkfurther out from the insert into the bordering maize genome with a final3′ flanking sequence, of 2346 bp.

In order to describe the TC1507 3′ flanking sequence, homology searchingwas done against the GenBank public databases using the Basic LocalAlignment Search Tool (BLAST). The BLAST program performs sequencesimilarity searching and is particularly useful for identifying homologsto an unknown sequence. In addition to searching the public databases,alignments were performed using SeqMan 4.05™, Martinez andNeedleman-Wunsch alignment algorithms (DNASTAR Inc.) to look forhomology between the TC1507 3′ flanking sequence and the PHI8999Atransgenic insert. The results of these homology searches are presentedin Table 1. The percent identity values indicate the percentage ofidentical matches across the length of the sequences analyzed. Theresults of similarity searching for the 3′ flanking sequence indicatehigh homology with three regions of maize chloroplast DNA, a 188 bpfragment of the pat gene, and 254 bp of DNA (Region 15, Table 1) with nosignificant homology. An additional 749 bp (Region 16) beyond Region 15(see Table 1) was also sequenced. No similarity searching results areavailable for Region 16.

PCR analysis on control and TC1507 genomic DNA determined that the 254bp sequence (Region 15, fragment of maize chloroplast “ORF241”) ispresent in the maize genome. The DNA sequence of Region 15 in the 3′flanking region is not unique to the 3′ flanking region of maize eventTC1507 but is also present in the unmodified control corn line. TheTC1507 3′ flanking sequence is presented in Example 8 and diagrammed inFIG. 1.

Example 8 Sequence of the Region 3′ to the End of the Full-Length InsertDNA in Maize Event TC1507 A Description of Each Region is in Table 1

Region 10 (SEQ ID NO: 38) Fragment of ORF25 Terminator (complement)

-   -   9016 CTCAC TCCGCTTGAT CTTGGCAAAG ATATTTGACG    -   9051 CATTTATTAG TATGTGTTAA TTTTCATTTG CAGTGCAGTA TTTTCTATTC    -   9101 GATCTTTATG TAATTCGTTA CAATTAATAA ATATTCAAAT CAGATTATTG    -   9151 ACTGTCATTT GTATCAAATC GTGTTTAATG GATATTTTTA TTATAATATT    -   9201 GATGATATCT CAATCAAAAC GTAGATAATA ATAATATTTA TTTAATATTT    -   9251 TTGCGTCGCA CAGTGAAAAT CTATATGAGA TTACAAAATA CCGACAACAT    -   9301 TATTTAAGAA ACATAGACAT TAACCCTGAG ACTGTTGGAC ATCAACGGGT    -   9351 AGATTCCTTC ATGCATAGCA CCTCATTCTT GGGGACAAAA GCACGGTTTG    -   9401 GCCGTTCCAT TGCTGCACGA ACGAGCTTTG CTATATCCTC GGGTTGGATC    -   9451 ATCTCATCAG GTCCAATCAA ATTTGTCCAA GAACTCATGT TAGTCGCAAC    -   9501 GAAACCGGGG CATATGTCGG GTATCTCGAG CTCGCGAAAG CTTGGCTGCA    -   9551 GGTCGACGGA TCCTT        Region 11 (SEQ ID NO: 39) Fragment of Maize Chloroplast rps12        rRNA Gene (Complement)    -   9566 CAACA AAAGGGTACC TGTACCCGAA ACCGACACAG    -   9601 GTGGGTAGGT AGAGAATACC TAGGGGCGCG AGACAACTCT CTCTAAGGAA    -   9651 CTCGGCAAAA TAGCCCCGTA ACTTCGGGAG AAGGGGTGCC CCC        Nucleotides 9694-9695 (CG) Connect Region 11 to Region 12.        Region 12 (SEQ ID NO: 40) Fragment of Maize Chloroplast Genome    -   9696 CTAAC    -   9701 AATAAACGAA TACGGTTTAT GTATGGATTC CGGTAAAATA CCGGTACTCG    -   9751 ATTTCATAAG AGTCGAATAG GAAGTTAAGA TGAGGGTGGT ATCATCATAA    -   9801 AAATGGAGTA GTATCCTAAA TTATACTAAT CCACGTATGA TATGTATGCC    -   9851 TTTCCTTATC AACCGGAAGT AGTGCAAAAA AAATTCTATA CTGCACTGCT    -   9901 CTCTTTTTAC TGAGAAATGC AAAAAAATAA AAGTGAAGTA AGGGTGCCCC    -   9951 ATAGATATTT GATCTTGCCT CCTGTCCCCC CCCCCCTTTT TTCATCAAAA    -   10001 ATTTCCATGA AAAAAGAAAA GATGAATTTG TCCATTCATT GAACCCTAGT    -   10051 TCGGGACTGA CGGGGCTCGA ACCCGCAGCT TCCGCCT        Region 13 (SEQ ID NO: 41) Fragment of pat Gene (Complement)    -   10088 GTT CCTAGCCTTC    -   10101 CAGGGCCCAG CGTAAGCAAT ACCAGCCACA GCACCCTCAA CCTCAGCAAC    -   10151 CAACCAAGGG TATCTATCTT GCAACCTCTC TAGATCATCAATCCACTCTT    -   10201 GTGGTGTTTG TGGCTCTGTC CTAAAGTTCA CTGTAGACGT CTCAATGTAA    -   10251 TGGTTAACGA TATCACAAAC CGCGG        Nucleotides 10276-10277 (AA) Connect Region 13 to Region 14.        Region 14 (SEQ ID NO: 42) Fragment of Maize Chloroplast ORF241        (Complement)    -   10278 CAC AAGAACGAAA GCACCTTTTC    -   10301 ATTCTTTCAT ATACTAGGGG TTTTTACTTG GAAAAGACAA TGTTCCATAC    -   10351 TAAAGGAT        Region 15 (SEQ ID NO: 43) Maize Genomic (No Significant        Homology)    -   10359 AG CTGCAGAAGC CGCCACCGTC TTGAGGACCT TCCGGGGAGC    -   10401 CAGACCGGTC GAACCGTGCC TCCACTTGCT AAGGAGAAAG GGAAAATCAG    -   10451 GGCCAGGACA TACGAAGGAG GAGCCAGAAC GAAGATATCC TAAGATACTT    -   10501 ACTCGCTCCG GGCCATGATC AATCATGCCT GTGGGGAGGT CTCTCGCACC    -   10551 TCGATCCATG AAGGTACCAC CGAGGTCTGC CCCGCCGCCG GCTTCGGTAC    -   10601 CGTCCTCGCC TT        Region 16 (SEQ ID NO: 44) Maize Genomic    -   10613 GGGCGCCC GAGGCACCCG GGGGATGGAC TGCCCAGGCG    -   10651 CAGCCACGAC GACCCAAGGA TCACCCTCCT GCGCAGTCGG CACGAGCAAT    -   10701 AGTTCTCGGG GAACAGGCAG CTTGGCCTGA CTCCCCGGGG TCACCTCAAC    -   10751 TACCTCGGCC GAGGGGTCAA GTACCCCCTC AGTCCGCCCC CGCTCTTCGG    -   10801 ACCGGGACCC CGACGTCCCG GCCCCGGATA CCGACGGCAC CAGCCCGCTC    -   10851 GGGGGCTGGC TTGACGACCC CTGGCCCAGC CTCAGATCTG GGCTGAGGCC    -   10901 GAGGCAGGCG GCCATGTCGT CGTCTTCATC ATCGTCTTCA TCATCGTCGT    -   10951 CGTCATCAGG CGTCTCCGGC GACGGCTCCC TTGGGAGCCC CTCCCTCTCC    -   11001 TGCCGACGAC GAAGCCTTTC CAAGGCATCC CGAGCCCACG TCCGCTCGTG    -   11051 GGCCCGAGCC TTCTTTGCGT CCTTCTTCTC CTTCCTCTTC TCCGCGGTGA    -   11101 CCCTCCGCGC AGCTCGGTCC ACCGCATCCT CCGGGACTGG TGGCAGGGAA    -   11151 GGCTTGTGAT GCCCTACCTC CTGGAGACAG ACGAAAAGTC TCAGCTATGA    -   11201 GAACCGAGGG CAATCTGACG CAAGAAGGAA GAAGGAGCGG ATACTCACCA    -   11251 GAGACACGCA CCCGCGATCG GGACGCATTA AGGGCTGGGA AAAAGTGCCG    -   11301 GCCTCTAATT TCGCTACCGT GCCGTCCACC CACCTGTGGA GGTCATCGAT    -   11351 GGGAAGGGGAA

Example 9 Confirmation of the Presence of Region 15 in the UnmodifiedControl Corn Line

PCR analysis was used to determine if the undescribed region of sequenceon the end of the 3′ flanking sequence (Region 15 in Table 1) is presentin the unmodified control corn line used for transformation to producemaize event TC1507 and thus represents a border with corn genomic DNA.Successful PCR amplification of Region 15 in both maize line TC1507 andthe unmodified Hi-II control corn line revealed that Region 15 wasindeed present in corn genomic DNA. Five different PCR analyses werecarried out on genomic DNA prepared from TC1507 and the unmodified Hi-IIcontrol corn line as outlined in Table 7 below using the primersequences shown in Table 8. Three reactions were designed to amplify DNAwithin Region 15 of the 3′ flanking region; Reaction L—producing a 175bp amplicon, Reaction M —producing a 134 bp amplicon, and ReactionN—producing a 107 bp amplicon. The expected amplicons were present inboth the unmodified control corn line and in maize line TC1507.Reactions J and K were designed as specific primer pairs for TC1507 thatwould span the end of the insert into the 3′ flanking region. InReaction J, the forward primer was located in the pat gene fragment onthe 3′ end of the full-length PHI8999A insert (Region 13 in Table 1) andthe reverse primer was located in the undefined Region 15. In Reaction Kthe forward primer was located in the chloroplast hypothetical proteingene on the 3′ end of the full-length insert (Region 14 in Table 1) andthe reverse primer was located in the undefined Region 15. Both ReactionJ and Reaction K produced an amplicon in maize line TC1507 and did notproduce an amplicon in the unmodified control corn line. The resultsindicate that both Reactions J and K are specific for the TC1507 event.

The PCR results indicate that the undescribed sequence (Region 15) ofthe 3′ flanking sequence of TC1507 is also present in genomic DNAisolated from the unmodified Hi-II control corn line. The DNA sequencesamplified in Reactions L, M, and N are not unique to the 3′ flankingregion of TC1507 but are also present in the unmodified control cornline.

TABLE 7 PCR reactions for sequence 3′ to the PHI8999A insert in maizeevent TC1507 Amplicon Amplicon present in Amplicon Region in TC1507present in maize line Reaction Size (bp) 3′ flanking sequence ControlTC1507 J 342 Region 13 No Yes (pat gene fragment) to Region 15 K 252Region 14 No Yes (chloroplast gene) to Region 15 L 175 Region 15 Yes YesM 134 Region 15 Yes Yes N 107 Region 15 Yes Yes

TABLE 8 PCR primers for sequence 3′ to the PHI8999A insert in maizeevent TC1507 Amplicon Primer Sequences Reaction Size (bp) Primer Pair 5′to 3′ J 342 SEQ ID NO:3 TGTGGTGTTTGTGGCTCTG TCCTAA SEQ ID NO:5GACCTCCCCACAGGCATGA TTGATC K 252 SEQ ID NO:4 AGCACCTTTTCATTCTTTC ATATACSEQ ID NO:5 GACCTCCCCACAGGCATGA TTGATC L 175 SEQ ID NO:16AAGCCGCCACCGTCTTGAG GACCTT SEQ ID NO:5 GACCTCCCCACAGGCATGA TTGATC M 134SEQ ID NO:17 GTCGAACCGTGCCTCCACT TGCTAA SEQ ID NO:5 GACCTCCCCACAGGCATGATTGATC N 107 SEQ ID NO:18 AGAAAGGGAAAATCAGGG CCAGGAC SEQ ID NO:5GACCTCCCCACAGGCATGA TTGATC

Example 10 PCR Primers

DNA event specific primer pairs were used to produce an amplicondiagnostic for TC1507. These event primer pairs include, but are notlimited to, SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 2 and SEQ ID NO:23; SEQ ID NO: 3 and SEQ ID NO: 5; and SEQ ID NO: 4 and SEQ ID NO: 5. Inaddition to these primer pairs, any primer pair derived from SEQ ID NO:26 and SEQ ID NO: 27 that when used in a DNA amplification reactionproduces a DNA amplicon diagnostic for TC1507 is an aspect of thepresent invention. The amplification conditions for this analysis areillustrated in Table 9, however, any modification of these methods thatuse DNA primers or complements thereof to produce an amplicon DNAmolecule diagnostic for TC1507 is within the ordinary skill of the art.The preferred amplification conditions for reactions utilizing the PCRprimers identified in SEQ ID NOS: 1, 2, and 23 are illustrated in Table10. In addition, control primer pairs, which include SEQ ID NOS: 10 and11; SEQ ID NOS: 10 and 12; SEQ ID NOS: 13 and 14; SEQ ID NOS: 14 and 15;SEQ ID NOS: 5 and 16; SEQ ID NOS: 5 and 17; and SEQ ID NOS: 5 and 18;for amplification of an endogenous corn gene are included as internalstandards for the reaction conditions. Also included are primer pairsthat will produce an amplicon in transgenic events containing a pat gene(SEQ ID NOS: 6 and 7; SEQ ID NOS: 8 and 9), and a primer pair that willproduce an amplicon in transgenic events containing a cry1F gene (SEQ IDNOS: 19 and 20).

The analysis of plant tissue DNA extracts to test for the presence ofthe TC1507 event should include a positive tissue DNA extract control (aDNA sample known to contain the transgenic sequences). A successfulamplification of the positive control demonstrates that the PCR was rununder conditions which allow for the amplification of target sequences.A negative, or wild-type, DNA extract control in which the template DNAprovided is either genomic DNA prepared from a non-transgenic plant, oris a non-TC1507 transgenic plant, should also be included. Additionallya negative control that contains no template corn DNA extract will be auseful gauge of the reagents and conditions used in the PCR protocol.

Additional DNA primer molecules of sufficient length can be selectedfrom SEQ ID NO: 26 and SEQ ID NO: 27 by those skilled in the art of DNAamplification methods, and conditions optimized for the production of anamplicon that may differ from the methods shown in Table 9 or Table 10but result in an amplicon diagnostic for event TC1507. The use of theseDNA primer sequences with modifications to the methods shown in Table 9and Table 10 are within the scope of the invention. The amplicon whereinat least one DNA primer molecule of sufficient length derived from SEQID NO: 26 and SEQ ID NO: 27 that is diagnostic for event TC1507 is anaspect of the invention. The amplicon wherein at least one DNA primer ofsufficient length derived from any of the genetic elements of PHI8999Athat is diagnostic for event TC1507 is an aspect of the invention. Theassay for the TC1507 amplicon can be performed by using a StratageneRobocycler, MJ Engine, Perkin-Elmer 9700, or Eppendorf MastercyclerGradient thermocycler, or by methods and apparatus known to thoseskilled in the art.

TABLE 9 PCR Conditions: Conditions: Kit used: Perkin-Elmer AmpliTAQ Goldkit Volume Component   5 μl template (10 ng/μl)   4 μl 2 μl each primer(10 μM)   2 μl 10× PCR Gold Buffer   2 μl 25 mM MgCl₂   2 μl 50× dNTP's(10 mM) 0.1 μl Amplitaq Gold Polymerase 4.9 μl H₂O  20 μl Total CyclingParameters GeneAmp ® PCR System 9700 9 min 92° C. 30 cycles: 94° C. 30sec 60° C. 30 sec 72° C. 1 min 7 min 72° C. Hold 4° C.

TABLE 10 PCR Conditions used with the Advantage ®-GC 2 Polymerase Mix:Conditions: Kit used: Advantage ®-GC 2 Polymerase Mix Volume Component  5 μl template (10 ng/μl)   5 μl 2.5 μl each primer (10 μM)   10 μl 5×GC2 Buffer   10 μl GC melt (1.0 M final conc.)  1.5 μl 50× dNTP's (10mM)  1.0 μl Advantage GC2 Polymerase 17.5 μl H₂O   50 μl Total CyclingParameters GeneAmp ® PCR System 9700 5 min 94° C. cycles: 94° C. 1 min60° C. 2 min 72° C. 3 min 7 min 72° C. Hold 4° C.

Having illustrated and described the principles of the presentinvention, it should be apparent to persons skilled in the art that theinvention can be modified in arrangement and detail without departingfrom such principles. We claim all modifications that are within thespirit and scope of the appended claims.

All publications and published patent documents cited in thisspecification are incorporated herein by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

1. An isolated DNA molecule consisting of the nucleotide sequenceidentified as SEQ ID NO: 26.